Discovery and Mechanistic Study of a Small Molecule Inhibitor for Motor Protein KIFC1

Abstract

Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a K i of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics.

KIFC1 is a novel potential therapeutic target for breast cancer

“…This is consistent with the finding from a recent study showing that KIFC1 expression is high in triple negative subtype of breast cancer. 15 Importantly, we also demonstrated for the first time that KIFC1 expression was higher in ER-negative and PR-negative breast cancer than in corresponding ER positive and PR positive breast cancer, but was not associated with HER2 status. Furthermore, in the present study we demonstrated that KIFC1 was expressed in breast cancer cell lines, but was absent in HMEC.…”

“…AZ82 and CW069 are 2 small molecule KIFC1 inhibitors reported so far, which inhibit microtubule-stimulated KIFC1 ATPase activity. 15,24 In the present study, we have identified PJ34 as a KIFC1 inhibitor by suppressing KIFC1 expressing in breast cancer cells. This finding supports the notion that PJ34 may be developed into an effective agent in treating breast cancer.…”

HSET overexpression fuels tumor progression via centrosome clustering-independent mechanisms in breast cancer patients

“…Docetaxel resistance in breast cancer is also suggested to be partly mediated by HSET [12]. In silico studies reveal that HSET expression is also higher in triple negative breast cancers compared to non-triple negative ones [13]. The differential dependence of cancer cells on HSET for viability and association of HSET expression with metastases-raise the tantalizing possibility that HSET may play a more important role in tumor progression than previously appreciated.…”

The Aspergillus nidulans bimC4 mutation provides an excellent tool for identification of kinesin-14 inhibitors

“…Importantly, as HSET is not essential for proliferation of normal cells with two centrosomes, HSET RNAi specifically kills cancer cells with extra centrosomes (Kwon et al, 2008). Small-molecule inhibitors of HSET/KIFC1 have been recently found using enzyme activity-based screens and iterative cycles of medicinal chemistry (Watts et al, 2013; Wu et al, 2013; Yang et al, 2014). However, their clinical efficacy has not been evaluated, and the possibility of off-target cytotoxicity has not been eliminated.…”

“…As first revealed in Saccharomyces cerevisiae , the microtubule minus-end-directed kinesin-14 antagonizes the plus-end-directed kinesin-5 during mitotic spindle assembly (Saunders and Hoyt, 1992). While loss of kinesin-5 causes a collapse of the bipolar spindle, leading to monopolar spindle formation, mitotic failure and cell death, loss of kinesin-14 attenuates these defects (Saunders and Hoyt, 1992; O’Connell et al, 1993; Mountain et al, 1999; Wu et al, 2013). Functions of these mitotic kinesins are highly conserved evolutionarily from fungi to humans.…”